Introduction

Lepr Rev (2001 ) 72, 1 79- 1 9 1

Response of Mycobacterium habana vaccine in

patients with lepromatous leprosy and their

household contacts. A pilot clinicai study

A . WAKHLU, S . P . S . GAUR * , G . P . KAU S HAL, ARVIND MISRA, O. P . A S THANA * & A . R . S IRCAR Department of Medicine, King George 's Medicai College, Lucknow,

u. P, India 226003

*Division of Clinicai and Experimental Medicine, Central Drug

Research Institute, P O Box 1 73, Chattar Manzil, Lucknow, u.p,

India 226001

Accepted for publication 5 April 2001

Summary Sing1e dose vaccination was carried out with Mycobacterium habana

vaccine, 3 1 1epromatous 1eprosy cases receiving 1 ·5 mg ( 1 '5mg=6'27 x 108 bacilli)

and 36 househo1d contacts randornly receiving 1 '5 , 2·0, 2'5 mg vaccine intradermal1y.

Duration of study was 1 8 weeks. Vaccination induced 1epromin conversion in 1 00%

of lepromatous leprosy cases and lepromin negative household contacts and aug­

mentation of lepromin reactivity in 1 00% of lepromin positive household contacts,

which was stable for the 1 5 weeks duration of folIow-up. The maximum augmenta­

tion in lepromin reactivity was obtained with 1 ·5 mg of vaccine, which is probably the

supramaximal dose. Overall, post-vaccination, those without prior BCG vaccination

scars showed higher mean values of lepromin augmentation. Local vaccination site

changes included induration, ulceration, itching, pain and uncomplicated regional

lymphadenopathy, alI of which remitted spontaneously by 1 5 weeks. Systemic side­

effects noted were pyrexia, ENL and jaundice, and were seen with no greater

frequency than that reported in other vaccine trials. Overall, systemic side-effects

were easily controlled and were not accompanied by clinically detectable nerve or ocular damage. The safety profile investigations revealed an increase in the mean values of Hb%, RBC count and PCV in household contacts and of PCV in lepromatous patients, post-vaccination. Alterations in the liver function tests were also observed in patients of lepromatous leprosy. Thus, M. habana vaccine appears to be useful in stimulating specific CMI against M. leprae as evidenced by increased lepromin reactivity.

Any vaccine potentially effective against Mycobacterium leprae should induce immune responsiveness to M. leprae. Various vaccine trials against leprosy have conc1usively shown

Correspondence to: A. Wakhlu, Department of ClinicaI Immunology, Sanjay Gandhi Postgraduate Institute of MedicaI Sciences, Rai Barielley Road, Lucknow, Uttar Pradesh, India (e-mai!: Anupam_wakhlu@hotmail.com)

0305-75 1 8/011064053+13 $ 1 .00 © Lepra 179

1 80 A. Wakhlu et aI.

changes in disease subtype, reduction in bacteriological and morphological indices, and protection in contacts of leprosy patients, postvaccination. I -7 The specific CMI against M. Ieprae and disease status correlate well with the degree of leprornin reactivity (delayed type hypersensitivity; DTH) of an individual . 1 .7-9

Mycobacterium habana (M. simiae serovar- l , TMC 5 1 35), an atypical mycobacterium belonging to Runyan group I, was obtained from the TMC Collection Center, Saranac Lake, New York, USA. M. habana vaccine was treated by gamma-irradiation of the culture at 300 kRad from a 60CO source. The immunoreactive fractions of the vaccine have been characterized by Chaturvedi et aI. 10 Mycobacterium habana protects rnice against infectious challenge with Mycobacterium Ieprae l l - 13 and induces leprornin positivity in monkeys . 14

The vaccine generated sufficient delayed-type hypersensitivity responses in rnice against M. Ieprae. 1 5 Various subunit components of M. habana, namely 65 kDa protein 16 and 23 kDa protein, 1 7 have been isolated and their putative role in immunity against M. Ieprae described. Thus, M. habana rnight prove to be useful as a potential vaccine against leprosy.

This study evaluated the efficacy (change in leprornin reactivity), safety and tolerance of M. habana vaccine in humans.

Materiais and methods

The entire preclinical data 18 on M. habana vaccine was subrnitted to the Drugs Controller of India and perrnission to undertake this clinical study was obtained. The study was also cleared by the Institutional Ethics Committee and informed written consent was obtained from alI the subjects before inclusion into the study. Total duration of study was 1 8 weeks.

S U B J E C T S ANO S T U O Y O E S I G N

Thirty-one lepromatous leprosy (LL) cases and 36 household contacts (HC) were vaccinated. Another group of 10 household contacts were included as controls; they were repeatedly leprornin tested but received no vaccine. Subjects under 1 2 years, pregnant women, those with major illnesses, and those who were taking immune modulatory drugs, were excluded from the study.

Patients with typical clinicaI features of Iepromatous Ieprosy, who were Ieprornin

negative, had numerous bacilli in their slit skin smears/skin biopsies and had typical features of Iepromatous leprosy on histopathology were included in the study, irrespective of their duration of treatment.

IndividuaIs from farnilies having at least one index case of lepromatous leprosy and who had been residing with the index case for at least 5 years were randomly included as household contacts for vaccination and as controls . They were included irrespective of their leprornin status.

F O L L O W - U P A N O E V A L U A T l O N O F L L C A S E S A N O H O U S E H O L O C O N T A C T S

After the initial pre-vaccination history and clinicaI exarnination (general and systernic) and initial leprornin test, the subject was vaccinated (day O), and closely followed for changes in their clinicaI status thereafter. Changes at the site of vaccination were noted as size of induration, ulceration, pain, itching, discharge and regional lymphadenopathy. LL cases were

M. habana vaccine in lepromatous leprosy patients and household contacts 1 8 1

assessed for their subjective feeling of well being and systemic reactions like pyrexia and erythema nodosum leprosum (ENL). Change in lepromin reactivity was read at +6 and + 1 5 weeks post-vaccination, i.e. 3 weeks after each lepromin injection. Safety profile investigations (Table 3) were done before vaccination and subsequently at +3 and + 15 weeks after vaccination.

During the trial, standard WHO multidrug therapy for multibacillary leprosy was continued and any other concomitant medication was avoided as far as possible.

LL cases were evaluated clinically using a body chart, the body being divided into head and neck, both upper limbs, both lower limbs, chest and abdomen and back and buttocks . Patients were c1inically exarnined for impairment of pain (pinprick), touch (wisp of cotton, tip of finger), temperature (test tubes with warm and cold water), vibration ( 1 28 Hz tuning fork) and reflexes in the distribution of the major nerves viz. great auricular, ulnar, median, lateral popliteal and posterior tibial nerves. Sensation was graded as normal, impaired (percent impairment from normal reference point) and absence of a particular modality. Corneal and conjunctival reflexes were tested. A detailed ophthalrnic evaluation was performed. Motor system was exarnined for nutrition, power (Standard MedicaI Research Council Scale, grade 0-5), reflexes and tone. Electromyography, nerve conduction velocity (n = 1 2) and slit lamp exarnination were done wherever possible (few cases on1y).

Each household contact was thoroughly evaluated for evidence of leprosy and only those who were c1inically free of the disease were inc1uded for vaccination or as controIs. Evaluation inc1uded a history for skin lesions, anaesthetic patches or other sensory symptoms and detailed exarnination for skin lesions, thickened nerves and sensory system exarnination.

V A C C I N E

Single dose vaccination was done with M. habana, a whole cell killed vaccine provided by Central Drug Research Institute, Lucknow. Each millilitre of vaccine contained 15 mg of wet weight of bacilli ( 1 ,5 mg = 6·27 x 108 AFB = 63 ·3 J-tg of protein). A single vaccine dose was injected intradermally into lefi upper deltoid region using calibrated tuberculin syringes. Local changes at vaccination site, systemic reactions and changes in lepromin reactivity were noted.

Contacts were randornly allocated to receive 1 ·5 (group I, n = 1 1 ), 2.0 (group lI, n = 1 3) and 2·5 (group lII, n = 1 2) mg vaccine, respectively, while lepromatous leprosy cases received 1 ·5 mg vaccine (group IV, n = 3 1 ) . Controls (group V, n = 1 0) received consecutive doses of lepromin but no vaccine.

L E P R O M I N

The leprornin (Mitsuda) test was performed using 0. 1 rnl of leprornin A, a suspension of killed M. leprae, obtained from GWL Hansen' s Disease Centre, Carville, Louisiana, 7072 1 , USA. Lepromin (0' 1 rnl) was injected intradermally at -3 , +3 and + 1 2 weeks (day O = day of vaccination) on the ventral surface of the right forearm at variable distances from each other and local response in the form of maximum diameter of nodule was read 3 weeks later. A response of 3 mm or greater was taken as a positive response.

B A C T E R I O L O G I C A L I N D I C E S ( B I ) ANO H I S T O P A T H O L O G Y

Slit skin smears were taken from six sites (two each from eyebrows, ear lobes and skin lesions). Slides were stained by the Ziehl-Neelsen method and BI was calculated according

1 82 A. Wakhlu et al.

to Ridley logarithmic scale. Skin punch biopsies were obtained for all LL cases from skin lesions.

Results

Lepromatous leprosy cases with disease duration ranging from 6 months to 3 years and were on standard WHO regimens for multibacillary leprosy at the time of vaccination. Patients had characteristic skin lesions at the time of vaccination, ranging from asymmetrical to bilaterally symmetrical shiny or erythematous macules, nodules and plaques with normal or impaired sensation and normal hair growth. Nerve thickening was seen in 60% of the cases. Impaired sensation (loss/impairment of temperature, pain, sweating and touch with relative preserva­tion of vibration and reftexes) in the distribution of ulnar, median and lateral popliteal nerves was noted in 20% of cases. No patient had facial nerve palsy, median nerve palsy, c1aw hand, foot drop, glove and stocking anesthesia or deformities. Disability grading (WHO) was grade O in 80% and grade 1 in the above-mentioned 20% of cases. Slit skin smears were performed in 24 of the 3 1 lepromatous leprosy patients, and revealed a median bacteriological index of 5 · 1 (range = 4,8-6,0). Skin biopsy and histopathological exarnination were performed in all 3 1 LL cases, which showed the characteristic histology of lepromatous leprosy in all cases.

The age and sex distribution among cases and contacts are shown in Table 1 . The age of contacts was significantly lower as compared to cases. The mean leprornin reaction in patients with lepromatous leprosy and various groups of household contacts is shown in Table 2.

L E P R O M I N C O N V E R S I O N A N D A U G M E N T E D L E P R O M I N R E A C T I V I T Y

Single dose vaccination induced leprornin conversion in 100% of lepromatous leprosy cases and leprornin negative household contacts (one contact in group I, 2 contacts in group 11, 1 contact in group I1I) and augmentation of leprornin reactivity in 1 00% of leprornin positive household contacts and was stable for the 1 5-week duration of follow-up. Post-Iepromin scar formation was observed in 65% lepromatous leprosy cases and 72-77% of household contacts (Table 2). The maximum increment in leprornin reactivity occurred in the initial 6 weeks after vaccination, subsequent to which the response plateaued; but the increments in lepromin reactivity continued up to 1 5 weeks post-vaccination, when the follow-up was

Table 1. Age and sex distribution of lepromatous leprosy cases and contacts (mean :t 5D)

Characteristic

Age

Sex

*p < 0.05 . **p < 0.0 1 . *** P < 0.00 1 .

Contacts (n = 36)

1 24.2 :t 8 . 1 * * * II 29.2 :t 14.2* m 30 :t 10. 1 *

Male = 58.3% (n = 2 1 ) Pemale = 41 .6% ( n = 1 5 )

Cases (n = 3 1 )

38.4 :t 14. 1

Male = 64.5% (n = 20) Pemale = 35.4% (n = 1 1 )

M. habana vaccine in lepromatous leprosy patients and household contacts 1 83

Table 2. Effect of M. habana vaccination on lepromin (Mitsuda) reaction

Dose of Leprornin (Mitsuda) test (mean ± SD, mm) NO. of vaccine

Group Clinical subjects (mg) Initial +6 weeks + 1 5 weeks

I II III IV V

HC 1 1 HC 1 3 HC 1 2 LL 3 1

Controls 1 0

H C = healthy household contact. LL = lepromatous leprosy cases.

1 ·5 7·0 ± 2·7 1 1 ·7 ± 2,5* 1 3-7 ± 1 ,9* 2·0 5·3 ± 2·8 10·6 ± 4, 1 * 1 1 ·9 ± 3·7* 2·5 5·4 ± 1 ·9 8 ·8 ± 2-4* 9·3 ± 3-4* 1 · 5 1 ·2 ± 1 -4 7·3 ± 3 ,3* 8·5 ± 4,4*

5·3 ± 1 ·8 5 ·5 ± 1 ·8 5 ·5 ± 1 · 8

Controls underwent consecutive leprornin testing but did not receive the vaccine. 'Percentage of patients with post-lepromin scar. *p < 0·05.

Post-Iepromin scar

( 1 5 weeks)*

72-2% 76·9% 75% 64·5%

terminated. Among the contacts of lepromatous leprosy cases, comparable degree of augmentation in leprornin reactivity post-vaccination was observed with 1 .5 mg and 2.0 mg vaccine, while a lesser degree of augmentation was seen with 2.5 mg vaccine. In 15% of cases, the size of the first 1eprornin test increased up to 1 5 weeks post-vaccination. Overall, post-vaccination, those without BCG vaccination scars showed higher mean values of leprornin augmentation (Figure 1 A-D). There was no relation between leprornin reactivity and the gender of the subject.

The control group of 10 household contacts of leprosy cases, of either sex, were given three leprornin injections consecutively (as per the schedule for those who received vaccination) but no vaccine. Initially, eight patients were leprornin positive (mean = 6 mm) and two were leprornin negative « 3 mm). The leprornin negative and six of the leprornin positive controls did not show any change in their consecutive leprornin reactivities. Two leprornin positive controls showed an increase of 1 ·0 mm in their subsequent leprornin reactivities (Table 2).

L O C A L S I T E C H A N G E S

The adrninistration of the vaccine did not produce any acute local reaction. The vaccine site remained quiescent for 7 -8 days. Mild to moderate pain and tendemess started at the local site by 7-8 days post-vaccination in almost all cases and persisted for about 6 weeks. Induration started by days 10- 1 2 and progressively increased till about day 1 6 and persisted thereafter for 7-8 weeks. The indurated area ulcerated by day 15- 1 8 post-vaccination and started healing by 8-9 weeks (Figures 2, 3). Healing was usually complete by week 15 post-vaccination (Figure 4). An indurated nodule was formed in 100% of cases. Mean diameter of nodule among contacts was 1 8 ·5 mm (range = 15-23 mm) and among LL cases was 15 ·4 mm (range = 8-20 mm). However, there was no correlation between the degree of induration and leprornin reactivity.

U1cers were formed in 100% of the cases and were punched out, with erythematous base and healthy looking granulation tissue. Mild serous or serosanguinous discharge was present. The mean diameter of u1cer among contacts was 7·5 mm (range = 4- 10 mm) and among LL cases was 6·5 mm (range = 4,5-9,0 mm). The u1cer required no treatment in any case except local cleaning. The degree of u1ceration and discornfort produced by vaccination was accepted by majority of subjects .

1 84 A. Wakhlu et aI.

L 1 6 E 1 4 P R 1 2 O M 1 0 I 8 N

6 I 4 N rn 2 rn O

I N ITIAL +6WEE K S +l5WE E K S

WEEKS AFrER VACCINA TION

L 1 6 E 1 4 P R 1 2 O M 1 0 I 8 N

6 I 4 N rn 2 rn O

IN ITIAL +6WE E K S +l5WEEKS

WEEKS AFrER VACCINATION

L 1 6 E 1 4 P R 1 2 O M 1 0 I 8 N

6

N 4

rn 2 rn O

I N ITIAL +6W E E K S + l 5 W E E K S

Figure 1. Lepromin reactivity (rnean :t SD) i n BCG scar positive and negative contacts and leprornatous leprosy (LL) cases, initially and after vaccination with Mycobacterium habana vaccine. (A) Eleven contacts, 1 ·5 rng vaccine; (B) 13 contacts, 2·0 rng vaccine; (C) 12 contacts, 2·5 rng vaccine; (D) 3 1 LL cases, 1 · 5 rng vaccine. Greater increase in lepromin reactivity was seen among BCG scar negative individuais (B & D).

M. habana vaccine in lepromatous leprosy patients and household contacts 1 85

L E p R O M I N

N m m

1 6 ,----------------------------, 1 4 +---------------------------� 1 2 +-------------------------1-� 10 +-------------+-4---------1-� ._�--� 8 +-------------+_ 6 +---------4 +-------/ 2 +---,--t---O

I N ITIAL +6W E E K S +l5WE E K S WEEKS AFTER VACCINA TION

Figure 1. continued.

Regional lymphadenopathy, 0,5- 1 ,5 em non-tender, non-matted and non-suppurative, was observed in a small percentage of cases (n = 1 1/67), which resolved spontaneously.

S Y S T E M I C R E A C T I O N S

Some patients (n = 42/67 ; 1 9/3 1 LL cases, 23/36 contacts) developed rnild to moderate pyrexia around days 3-4, which resolved in 7 - 1 0 days with antipyretics.

Twenty-six percent (n = 8/3 1 ) of the lepromatous leprosy cases developed ENL at 6-9 weeks post-vaccination. Of these, 25% (n = 2/8) had experienced previous episodes of ENL (ranging from one to three episodes). Patients developed rnild to moderate pyrexia, aches,

Figure 2. Vaccination site changes (induration and ulceration) 3 weeks afier M. habana vaccination.

1 86 A. Wakhlu et aI.

Figure 3. Vaccination site changes (induration and u1ceration) 9 weeks after M. habana vaccination.

arthralgias (but no arthritis) and an increase in the size and number of cutaneous lesions in the forro of tender, erythematous macules, nodules and plaques. The nerves became more tender on palpation, reftexes were exaggerated almost universally; however, there was no clinically manifest increase in or new development of hypoaesthesia or anaesthesia. There was no

Figure 4. Vaccination site changes (healed scar) 15 weeks after M. habana vaccination.

M. babana vaccine in lepromatous leprosy patients and household contacts 1 87

Table 3. Mean values of safety profile investigations initially and after vaccination with M. habana vaccine in lepromatous leprosy cases (LL) and household contacts (HC) (mean :!: SD)

Investigation Group Initial +3 weeks + 15 weeks

Hb % HC 1 1 ·8 :!: 1 ·7 l 1 'O :!: 1 ·7 1 2'2 :!: 1 -4 LL l 1 'O :!: 1 ·8 1 2'5 :!: 1 · 1 * 1 1 ·7 :!: 1 ·7

TLC (per mm3) HC 743 1 -4 :!: 38·7 7758 · 1 :!: 2018 ·3 7297 :!: 894·2 LL 7520 :!: 1452 7 1 70·6 :!: 932·5 7384·2 :!: 2018 ·3

Platelets (per mm 3) HC 2·6 :!: 0-4 2·7 :!: 0·3 2·7 :!: 0·2 LL 2'6 :!: 0·2 2'7 :!: 0·4 2'7 :!: 0·2

RBC (per mm3) HC 3-9 :!: 0-4 3·7 :!: 0·5 3'9 :!: 0·4 LL 3 -4 :!: 0·5 4· 1 :!: 0,3* 3 ·8 :!: 0·5

ESR (mm 1 st h) HC 9·5 :!: 6·3 10-4 :!: 6· 1 9·6 :!: 4·5 LL 9·6 :!: 5·6 8 ·70 :!: 6·9 1O'0 :!: 5·3

PCV (cc%) HC 36·5 :!: 5·5 33·6 :!: 5 ·0 38·2 :!: 5·6 LL 34·8 :!: 4·6 38·4 :!: 3 ,6** 36·3 :!: 4·5**

Blood urea (mmolfl) HC 8·6 :!: 2·8 9·8 :!: 4·3 8·3 :!: 1 ·4 LL 8'7 :!: 2·6 8·6 :!: H 9·4 :!: 2·0

Serurn creatinine (/Lmolfl) HC 70·7 :!: 8·8 70·7 :!: 8·8 70·7 :!: 8·8 LL 79·5 :!: 8·8 6 1 ·8 :!: 8·8 79·5 :!: 8·8

Random blood sugar (mmolfl) HC 5·3 :!: 2·7 5·2 :!: 1 ·2 5'6 :!: 1 ·4 LL 5 '0 :!: 1 ·5 5·7 :!: 3 ·0 5 - 1 :!: 1 ·0

Serum bilirubin (/Lmolfl) HC 1 3 ·6 :!: 3·4 1 3 -6 :!: 3-4 1 3 -6 :!: 3 -4 LL 15 ·3 :!: 8·5 1 3 ·6 :!: 3·4 17 · 1 :!: 13 -6

SGOT (IU1l) HC 24· 1 :!: 6 · 1 24·7 :!: 8·4 25·9 :!: 14·4 LL 23·7 :!: 4·9 25·6 :!: 9·9 29·7 :!: 8,9*

SGPT (IUIl) HC 33·3 :!: 2 1 ·8 30·7 :!: 1 8 · 1 3 1 ·5 :!: 20·4 LL 35·9 :!: 26·5 27-4 :!: 1 2 · 1 40·2 :!: 35·3

Serum aIkaline HC 1 1 3 :!: 63 99 :!: 52 99 :!: 46 phosphatase (IUIl) LL 97 :!: 35 1 12 :!: 46 1 16 :!: 49*

Serum protein (gIl) HC 82 :!: 8 79 :!: 7 82 :!: 10 LL 79 :!: 6 76 :!: 5* 77 :!: 6*

Serum alburnin (gll) HC 45 :!: 4 44 :!: 4 46 :!: 3 LL 44 :!: 4 46 :!: 3 42 :!: 4

Serum uric acid (/Lmolfl) HC 404·4 :!: 1 1 8·9 422·3 :!: 83·2 404·4 :!: 95- 1 LL 386·6 :!: 65-4 446· 1 :!: 1 90·3 422·3 :!: 89·2

Serum cholesterol (mmol/1) HC 3 ·8 ± 0·9 3 ·5 ± 0·9 3 ·6 ± 0·8 LL 3·5 ± 0·8 4·2 :!: 0·8 3·5 :!: 0-4

Serum triglyceride (mmol/1) HC 1 ·7 :!: 0·8 1 ·5 :!: 0·7 1 ·4 ± 0·5 LL 1 ·9 :!: 1 · 2 1 ·9 :!: 0·8 1 ·7 :!: 0·7

Serum HDL (mmolfl) HC 0·9 :!: 0·2 0·9 :!: 0· 1 0·9 :!: 0 · 1 LL 0·9 :!: 0 · 1 0 ·9 :!: 0· 1 0·9 :!: 0· 1

*p < 0·05. **p < 0·0 1 . * * *p <0,001 . Hb, haemoglobin; TLC, total leukocyte count; RBC, red blood cell count; ESR, erythrocyte sedimentation rate;

PCV, packed cell volume.

1 88 A. Wakhlu et al .

weakness or paralysis of muscle groups and no muscle wasting. These episodes were not associated with any organomegaly, cardiac or pulmonary abnormalities. There was no rhinitis, epistaxis or epididymo-orchitis . There was no swelling, discharge, or pain in the eye. Comeal reflexes were intact in alI the patients. The episodes of ENL were easily controlIed with antipyretics and non-steroidal anti-inflammatory drugs, requiring cortico­steroids in only two cases. Duration of ENL was 3-4 weeks except in two cases where it lasted for 7 -8 weeks. OveralI, the systernic side effects were easily controlIed and were not accompanied clinicalIy by organ, nerve, or ocular damage.

Two patients, both of whom had ENL, developed concornitant clinicaI jaundice 4 weeks post-vaccination. In both cases, patients had been taking MDT for more than 6 months. The jaundice was insidious in onset, associated with a prodrome of nausea, vorniting and malaise 1 week prior to clinicaI detection. Both the patients had 3-4 cm tender, soft hepatomegaly with no splenomegaly. Serum bilirubin (patient 1 , 3 ·5 mg% ; patient 2, 4·5 mg%), SGOT (patient 1 , 150 IUIl ; patient 2, 2 10 IUIl) and SGPT (patient 1 , 450 IUIl; patient 2, 600 IUIl) were elevated with marginalIy raised serum alkaline phosphatase (patient 1 , 140 IUIl; patient 2, 1 60 IUIl). Ultrasound revealed hepatomegaly with a rnild decrease in echogenicity and contracted galIbladder in both the cases. Rifampicin, dapsone and clofazirnine were stopped on account of possible hepatotoxicity. Viral markers for HBsAg and anti-HCV were negative. Both patients were conservatively managed and recovered completely by the end of 3 weeks after clinical detection, after which drug therapy was re-started, without subsequent re-appearance of jaundice tilI the end of folIow-up.

There was complete absence of nerve palsies both among cases and contacts, post-vaccination and no new sensory or motor loss was detected in these patients.

One lepromatous patient presented with bilateral pitting pedal oedema 3 weeks after vaccination, non-commensurate with his serum protein and alburnin status, which had resolved completely by the end of the study.

Two patients presented 4-6 weeks post-vaccination with papulonodular eruptions alI over the body. No features suggestive of ENL were present. The rashes subsided on their own at final folIow-up.

I N V E S T I G A T I O N S

There was an increase in the mean values of Hb%, RBC count and PCV in household contacts and of PCV in lepromatous patients, post-vaccination. Alterations in the liver function tests were also observed in patients of lepromatous leprosy (Table 3), especially in the two who developed clinicaI jaundice and ENL. Slit lamp exarnination could not be performed due to constraint of resources. Other patients who developed ENL had no proteinuria and the liver and renal function tests were unremarkable.

Discussion

Leprosy has a very long incubation period, and years of observation will therefore be required to measure the efficacy of a vaccine in terms of its capacity to lower incidence rates. As a first step, it is essential to show that a 'candidate' vaccine is able to induce persistent changes in immunity as evidenced by changes in the Mitsuda leprornin reaction. A vailable clinicaI, laboratory and experimental evidence clearly show that the late leprornin (Mitsuda) reaction

M. habana vaccine in lepromatous leprosy patients and household contacts 1 89

is c10sely linked to the immune status of the host against M. leprae. Early work by Dharrnendra and Chatterjee8 has shown that lepromin-negative individuaIs in endemic areas mn a very high risk of contracting the multibacillary forms of the disease.

One hundred percent lepromin conversion or augmentation, which was observed in the present study with a single dose of vaccine, has been rarely reported with adrninistration of other vaccines . 1 -5 • 19 In the present study, the maximum change in lepromin reactivity was observed in the first 6 weeks post-vaccination, after which the degree of rise, although continuing for 1 5 weeks, plateaued off. Further follow-up will be required to ascertain whether this gradual rise continues, stabilizes or steadily declines. The maximum increment in lepromin reactivity with other vaccines has been variably described from 1 month onwards; most have described peak values at 8-9 months post-vaccination with stability for 3-5 years of follow-up.

The present study has demonstrated that the maximum lepromin augmentation or conversion was obtained with the smallest dose of 1 ·5 mg vaccine. That 1 ·5 mg vaccine is probably the maximum tolerated dose and requires reduction is further suggested by the constellation of reduced responses with higher doses of 2·5 mg and higher incidence of ENL reaction.

The diminished responses seen with higher doses of vaccine can be explained by either suppression of immature B-cells by antigen excess or the suppression of cell mediated immunity by 'blocking' immune complexes.

The degree of lepromin conversion or augmentation in lepromin reactivity was consist­ently less among BCG scar positive individuals. This is contrary to other leprosy vaccine trials, which show a higher degree of augmentation among BCG vaccinated individuals. 1

The phenomenon of augmentation in the nodule size of prevaccination lepromin after vaccination has also been reported by Deo et al .2 and probably refiects continuing delayed reactivity at site of first lepromin test; the c1earance of M. leprae is markedly retarded in lepromatous leprosy patients as shown by Convit et al.20

Sixty to 75% of all subjects in the present study developed post-Iepromin scars, which have been proposed as indicators of stable changes in cell mediated immunity by Dharmendra9 and Walters?1

A control group of unvaccinated, healthy household contacts who were repeatedly lepromin tested did not exhibit any change in subsequent lepromin reactivity, indicating that the conversion was not merely a consequence of the previous lepromin test and that lepromin did not act as a mini-vaccine. It has also been demonstrated that M. leprae bacilli (arrnadillo grown), at 4 times the concentration contained in lepromin, failed to induce lepromin conversion?2 Household contacts were chosen as controls because they had a greater chance of reacting to lepromin as compared to LL cases who are anergic to lepromin (lepromin negative) . Since repeated lepromin testing did not alter the lepromin reactivity of lepromin positive or lepromin negative household contacts, lepromin negative LL cases were not inc1uded as controls subsequently.

The local vaccination site changes observed with M. habana vaccination are similar to those observed with various other vaccines.2,4,6 The large induration and u1ceration occurring in a number of vaccinated subjects may, by itself, not be considered as an unacceptable side effect of the vaccine, given that most people have accepted both BCG and smallpox vaccines. None of the local site changes bore any correlation with lepromin conversion in the present study.

Zaheer et aI. observed type I upgrading reactions in 45 .2% of their vaccinated

190 A. Wakhlu et alo

lepromatous leprosy cases.4 We did not observe type I reactions during our study, which could be due to the short duration of our study or because only a single dose of vaccine was given in our trial as opposed to the other vaccine trials.4

Twenty-six percent of lepromatous leprosy patients in the present study developed type 2 reactions in variable time duration of 6-9 weeks post-vaccination. Development of ENL after vaccination has been described by Deo et aI. with ICRC vaccine, to occur 3-4 weeks after vaccination in 42·2% cases with high bacteriological index.2 ENL after vaccination has also been described by Zaheer et ai. with Mycobacterium w vaccine, to occur in 56·6% of lepromatous leprosy cases, while unvaccinated patients taken as controls developed ENL in 60·2% cases.4

Two cases of ENL in the present study developed jaundice; The jaundice could have been a result of concomitant viral hepatitis or due to ENL. Convit et aI. have observed two cases of hepatitis with jaundice in borderline leprosy cases with reversal phenomenon post vaccination.6

Two lepromatous patients in the present study developed a papulonodular eruption 4-6 weeks after vaccination. This has been previously reported by Convit et al. ,6 who demon­strated tuberculoid histology of the papules. The rash was seen to subside on its own at subsequent follow-ups.

The changes in safety profile investigations did not reveal any abnormality that could directly be attributed to the vaccine except for changes in liver function tests in lepromatous leprosy cases which could also be due to concomitant multi drug therapy regimen or due to the nature of the disease itself.

Thus, M. habana vaccine appears to be useful in stimulating specific cell mediated immunity against M. leprae as evidenced by lepromin conversion or augmentation of reactivity observed in the present study. Further, the vaccine, by virtue of minimal, well tolerated side-effects and good acceptability among the trial population, may be deemed tolerable.

References

[ Deo MG. Anti leprosy vaccines - field trials and future prospects. Ind J Lepr, 1984; 56: 764-775. 2 Deo MG et ai. Antileprosy potentials of the ICRC vaccine. A study in patients and healthy volunteers. Int J Lepr,

1983; 51: 540-549. 3 Karonga Prevention Trial Group. Randomized control trial of single BCG, repeated BCG or combined BCG and

killed M. leprae vaccine for prevention of leprosy and tuberculosis in Malawi. Lancet, 1996; 348: 17-24. 4 Zaheer SA, Beena KR, Kar HK et ai: Addition of immunotherapy with Mycobacterium w vaccine to multidrug

therapy benefits multibacillary leprosy patients. Vaccine, 1995; 13: 1 102- 1 1 10. 5 Zaheer SA, Mukhetjee R, Ramkumar B et aI. Combined multidrug and Mycobacterium w vaccine therapy in

patients with multibacillary leprosy. J Infect Dis, 1993; 167: 401 -410. 6 Convit J, Aranzazu M, UJrich M et aI. Immunotherapy with a mixture of M. leprae and BCG in different forms of

leprosy and in Mitsuda negative contacts. Int J Lepr, 1 982; 50: 415-424. 7 Bloom BR. Rationales for vaccines against leprosy. Int J Lepr, 1983; 51: 505-509. 8 Dharmendra, Chattetjee KR. Prognostic value of the lepromin test in contacts of leprosy cases. Int J Lepr, 1956;

24: 3 15-3 18 . 9 Dharmendra, Chatterjee KR. Prognostic value of lepromin in contacts of leprosy cases. Lepr Ind, 1955; 27:

149- 152. [O Chaturvedi V, Singh MB, Sinha S. Immunoreactive antigens of a candidate leprosy vaccine: Mycobacterium

habana. Lepr Rev, 1995; 66: 3 1 -38. [ [ Singh NB et aI . Immunological potential of cultivable mycobacterial strain M. habana against leprosy bacillus in

mouse footpad. Ind J Lepr, 1985; 57: 278-28 1 . [ 2 Gupta HP, Singh NB, Ashok Kumar. Containrnent of Mycobacterium leprae multiplication i n foot pad of

M. habana vaccine in lepromatous leprosy patients and household contacts 1 9 1

M. habana vaccinated animals. Biol Mem, 1987; 13: 1 74- 178. 1 3 Singh NB et ai. Vaccination of mice against M. leprae infection. Infect Immun, 1989; 57: 653-655 . 14 Singh NB et ai. Induction of lepromin positivity in monkeys by a candidate antileprosy vaccine, Mycobacterillm

habana. Int J Lepr Other Mycobact Dis, 1 99 1 ; 59: 3 17-320. 1 5 Singh NB et aI. Lymphostimulatory and delayed-type hypersensitivity responses to a candidate leprosy vaccine

strain: Mycobacterillm habana. Lepr Rev, 1 997; 68: 1 25- 1 30. 16 Singh NB et aI. The 65 kDa protein of M. habana and its putative role in immunity against experimental

tuberculosis. Immunol Cell Biol, 1 995 ; 73: 372-376. 1 7 Bisht D et ai. A major T -cell inducing cytosolic 23 kDa protein antigen of the vaccine candidate M. habana is

superoxide dismutase. Microbiology, 1 996; 142: 1 375- 1 383. 1 8 Data on Mycobacterillm habana (antileprosy) vaccine (Dossier) December 1987, Central Drug Research Institute,

Chattar Manzil, Lucknow. 19 Anantharaman DS, Valishayee RS, Gupte MD et ai. Post vaccination sensitization with ICRC vaccine. lnd J Lepr,

1996; 68: 1 67- 1 74. 20 Convit JA, Pindari ME. Elimination of M. leprae subsequent to in vivo activation of macrophages in lepromatous .

leprosy by other mycobacteria. Clin Exp Immunol, 1974; 17: 261 -265. 2 1 Walter J, Tamodong CT, Bechelli PG et aI. Notes on some observations about the post lepromin scar. Lepr Rev,

1977; 8: 1 69- 1 74. 22 Gill HK, Mustafa AS, Godal T. Induction of DTH in human volunteers immunized with a candidate leprosy

vaccine consisting of killed M. leprae. Buli WHO, 1 986; 64: 1 2 1 - 1 26.